Microbial collagenases (EC 3.4.24.3) are metalloproteinases which degrade helical regions of native collagen to small fragments. Preferred cleavage is -Gly in the sequence -Pro-Xaa-Gly-Pro-. Six main forms grouped in two classes have been isolated from Clostridium histolyticum that are immunologically cross-reactive but possess different sequences and different specificities. Other variants have been isolated from Bacillus cereus, Empedobacter collagenolyticum, Pseudomonas marinoglutinosa, and species of Vibrio and Streptomyces. 
Collagenases have a role in destroying extracellular structures in the pathogenesis of bacteria such as Clostridium histolyticum. Thus, they are exotoxins and act as virulence factors, e.g., by facilitating the spread of gas gangrene. Collagenases normally target the connective tissue in muscle cells and other body organs. Owing to the potent hydrolytic activity toward connective tissue, collagenases and other proteinases such as thermolysin are used for tissue dissociation in vitro.
The collagenases produced by Clostridium histolyticum were the first collagenases to be discovered and characterized. The culture filtrate of Clostridium histolyticum contains a mixture of collagenases and other proteinases. Six main collagenases with molecular masses ranging from 68 to 130 kDa have been purified to homogeneity and have been designated as type I and type II collagenases (both classes of proteins are herein also referred to collectively as the “collagenase proteins”). Said clostridial collagenases contained approximately one of zinc per protein chain, whereby the zinc atom appears to be essential for enzymatic activity.
At a technical scale, clostridial collagenases are isolated from the culture filtrate of Clostridium histolyticum. The crude preparations contain the collagenases as well as a brown pigment, clostripain (clostridiopeptidase B), an aminopeptidase and several neutral proteinases. Crude preparations can serve as a source for the purification of the individual type I and II collagenases, but the purification scheme is quite long.
The presence of the cysteine protease clostripain in the mixture poses a significant technical problem since this peptidase progressively hydrolyzes the collagenases. In this regard collagenase of the type I appears to be more sensitive to proteolytic attack by clostripain than type II collagenase. When purifying collagenases from C. histolyticum culture filtrate or liquid culture supernatant it is therefore desirable to separate clostripain at an early step to minimize losses.
Bond, M., D., Van Wart, H., E., (Biochemistry 23 (1984) 3077-3085) disclose a chromatography-based purification scheme which starts with a crude enzyme preparation. In the first step the crude enzyme was chromatographed over hydroxylapatite. The mechanism of hydroxylapatite chromatography is also known as “mixed-mode” ion exchange. It involves nonspecific interactions between positively charged calcium ions and negatively charged phosphate ions on the stationary phase hydroxylapatite resin with protein negatively charged carboxyl groups and positively charged amino groups. For elution, a buffer with increasing phosphate concentration is typically used. According to Bond, M., D., Van Wart, H., E. (supra) three fractions were eluted with a potassium phosphate gradient. The first fraction contained the majority of the pigment and the third fraction contained 95% of collagenase activity. The third fraction was further subjected to gel filtration chromatography on a SEPHACRYL S200 column (GE Healthcare Bio-Sciences AB), followed by affinity chromatography over L-arginine-AFFI-GEL 202 (Bio-Rad Laboratories, Inc.). These steps combined and in the order as disclosed served to remove the brown pigment and the majority of the contaminating proteinases active against casein, benzoyl-L-arginine ethyl ester, and elastin. Reactive Red 120 dye ligand chromatography subdivided the type I and type II collagenases.
WO 2003/004628 discloses a purification scheme in which C. histolyticum culture supernatant is subsequently chromatographed over (1) hydroxylapatite, (2) an anion exchanger resin, and (3) a cation exchanger resin. The third fraction eluted from the hydroxylapatite column represented the collagenase I/II fraction but also contained clostripain. The anion exchanger was eluted to separate in two fractions collagenase I and collagenase II. However, both fractions still contained clostripain. The collagenases were separated from clostripain using the cation exchanger.
The methods described above represent different chromatographic approaches aiming to eliminate the undesired clostripain from the collagenases.
WO 2007/089851 discloses an assessment of precipitation with ammonium sulfate as a primary recovery step for the collagenase proteins from culture filtrate which contained significant amounts of other proteases such as clostripain. Accordingly, ammonium sulfate was added to the filtrate, initially at concentrations between 100 and 400 g/l and furthermore at concentrations between 400 and 520 g/l. Recovery of collagenase by precipitation was found to be significant at 400 g/l and the pellet generated using this concentration was found to be the easiest to resuspend. Furthermore, concentrations higher than 400 g/l apparently resulted in a very similar recovery with respect to the collagenase proteins.
In order to produce collagenases at a larger scale, WO 2007/089851 discloses the combination of (1) a specially chosen strain of C. histolyticum, (2) a fermentation process with growth conditions to optimize production of collagenases while aiming to reduce the production of clostripain, and (3) a purification scheme for collagenase I/II.
Both elements, the choice of the C. histolyticum strain and the liquid fermentation medium contribute to a reduction of clostripain in the raw material used for the purification process. Accordingly, it is noted that the conditions disclosed in WO 2007/089851 are such that the purification scheme is based on a raw preparation with a specific clostripain activity of between 0.7 and 5.5 U per mg total collagenase. According to the disclosure, these values also reflect the clostripain vs. Collagenase ratio in the culture supernatant since no noticeable separation of clostripain was found as an effect of ammonium sulfate precipitation. The comparably low amount of clostripain (between 0.7 and 5.5 U per mg total collagenase) appears to be an effect of the choice of peptones in the fermentation broth. Particularly, a porcine-derived peptone is used to provide this effect.
A raw preparation of collagenase containing fermentation product was obtained by way of ammonium sulfate precipitation from the fermentation broth.
The purification scheme of WO 2007/089851 starts with filtration of the liquid fermentation batch. Following addition of ammonium sulfate to the filtrate and removal of unsoluble matter, hydrophobic interaction chromatography is performed in the presence of ammonium sulfate in the liquid phase. Leupeptin, a well-known (reversible) inhibitor of clostripain and other serin and cystein proteases, was added to the eluted fractions comprising the collagenases, indicating that no complete separation of clostripain and/or other proteases was achieved. Leupeptin is removed during a later step in the purification process.
It is also noted that the final purified collagenase I and collagenase II preparations additionally contain N-terminally cleaved degradation products and for collagenase I also C-terminally cleaved degradation products.
The extensive use of leupeptin in the purification method of WO 2007/089851 indicates that even after hydrophobic interaction chromatography a significant residual amount of undesired proteolytic activity is present in the eluted fractions comprising the collagenases. Such residual proteases are particularly disadvantageous because they rapidly degrade the desired collagenases, whereby collagenase type I is a particularly sensitive target.
In contrast to the state of the art, the present invention is based on a culture filtrate or supernatant, in which the fermentation broth for C. histolyticum culture is free of any mammalian-derived component (such as a peptone) and in which the specific clostripain activity in the filtrate or supernatant is between about 10 and about 200 U per mg total collagenase, preferred between about 50 and about 200 U per mg total collagenase, more preferred between about 75 and about 200 U per mg total collagenase, even more preferred between about 100 and about 200 U per mg total collagenase.
The inventors have surprisingly found that a precipitation step with ammonium sulfate ((NH4)2SO4) prior to hydrophobic interaction chromatography allows to obtain a stabilized intermediate product comprising the desired type I and type II collagenases. Upon recovering of the precipitate and redissolving the same, hydrophobic interaction chromatography can be performed to reduce the activity of clostripain by the factor of about 100 to about 150 and the activity of trypsin by the factor of 100-400 in the resulting pooled eluate comprising the collagenases, whereby in the pooled eluate 80-95% of the combined collagenases I and II are present, as compared to the amounts present in the redissolved precipitate. As a result, after hydrophobic interaction chromatography the mixture of collagenases I and II is substantially stable in the absence of any protease inhibitor (e.g., leupeptin). That is to say, under the conditions of the invention none or only minimal proteolytic degradation of collagenase I or II is detectable. Importantly, removal of concomitant non-collagenase proteases in the preparation is so effective that further purification of collagenases I and II leads to products having an exceptional purity and even lacking N-terminal degradation.